Cephalosporin C acylase is an enzyme that converts cephalosporin C to 7-amino-cephalosporanic acid (7-ACA), an intermediate for the preparation of a large number of semisynthetic cephalosporins.
Even if 7-ACA can be obtained from cephalosporin C by chemical synthesis, enzymatic methods are preferred in that they are more environment-friendly and less costly.
Conventional enzymatic procedures for the conversion of cephalosporin C in 7-ACA require two different enzymes, a D-amino acid oxidase (DAAO) and a glutaryl acylase. DAAO transforms cephalosporin C into α-Keto-adipoyl-7ACA with concomitant production of hydrogen peroxide. α-Keto-adipoyl-7ACA is then oxidized to glutaryl-7-ACA with hydrogen peroxide (produced by oxidative deamination and/or added to the reaction medium), thereafter glutaryl acylase hydrolyses glutaryl-7-ACA to 7-ACA. The conventional process requires two separate enzymatic reactors, and the presence or addition of hydrogen peroxide can inactivate the immobilized enzymes, which impairs the plant and increases costs.
Attempts have been made to develop enzymes able to directly hydrolyse cephalosporin C to 7-ACA. Three known enzymes, referred to as SE83, N176 and V22, isolated from Pseudomonas strains, possess this ability (Journal of Fermentation and Bioengineering Vol. 72(4), 232-243, 1991), but their acylase activity is higher on glutaryl-7ACA than on cephalosporin C.
Mutants and recombinant DNA methods for their preparation have been disclosed (U.S. Pat. No. 5,320,948, EP 475652, EP 558241, U.S. Pat. No. 5,804,429) to improve the characteristics of these enzymes, in particular specificity, stability and activity.
However, there is still the need for enzymes with improved cephalosporin C acylase activity, in terms of kinetics, stability, activity and specificity and which can be expressed in high amounts.